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Turning on Genes with The Blues

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Scientists have developed a light-controlled spatiotemporal system for controlling the expression of target genes within cells in a population, which they claim is tunable, reversible, repeatable, and can be used to target a diverse range of DNA sequences. The new system, which its Duke University developers Lauren R. Polstein, Ph.D., and Charles A. Gersbach, Ph.D., have called Litez (Light-induced transcription using engineered zinc finger proteins), combines heterodimerizing, light-sensitive proteins derived from the flowering plant Arabidopsis thaliana, and a zinc finger protein (ZFP) transcription factor. In essence, when cells carrying the Litez constructs are illuminated with blue light, the zinc finger protein is activated and turns on transcripton of its target gene.

In practical terms the platform uses two fusion proteins. The first comprises one of the two Arabidopsis proteins fused to three repeats of the transcriptional activation domain VP16. The other comprises the other light-sensitive protein fused with a zinc finger protein and (if required) a fluorescent reporter. This second protein effectively localizes the ZFP to the target gene. When cells containing both these constructs are illuminated with blue light, the two Arabidopsis proteins dimerize, bringing the transcription activation domain carried by one of the proteins over to the gene that has already been targeted with the ZFP-containing construct, and activating gene transcription.

The Duke researchers tested their technology in colonies of cells growing in vitro. When a mask or stencil was placed over the colonies and the blue light switched on, the cells subjected to the light switched on the target gene expression, and the reporter molecules attached to the ZFP lit up. Depending on the stencil used to cover the cell colonies, they generated patterns of fluorescence in the cell colonies including a smiley face, and even a letter D, for ‘Duke’.

Drs. Polstein and Gersbach point out that, while light-inducible gene expression isn’t a new concept, previous systems have relied on common DNA-binding domains, which means all the regulated transgenes have to contain the upstream binding sequence that matches that particular DNA-binding protein. In contrast, Litez allows almost any sequence to be targeted with engineered ZFPs, providing the freedom to use diverse promotor sequences to control a combination of transgenes.

Describing Litez in the Journal of the American Chemical Society, the investigators say the system could be harnessed practically for new approaches to gene therapy, synthetic biology, biopharmaceutical production, and tissue engineering. "Spatial patterning of gene expression via light allows for unique applications of inducible gene regulatory systems," they claim.

“This approach can be applied to the field of tissue engineering to create complex constructs that recapitulate the morphology and functionality of natural tissues. Spatial activation of key morphogenetic factors could be used to precisely engineer tissues patterned with multiple cell types. As a result, engineered tissues will more closely mimic native tissues, increasing the probability of implant survival and improving tissue functionality. The spatial control provided by LITEZ, combined with reversible and repeatable gene activation, will also enable novel basic science studies of gene function, gene regulation, and cell–cell interactions”

The Duke researchers describe the Litez technology in a paper titled “Light-Inducible Spatiotemporal Control of Gene Activation by Customizable Zinc Finger Transcription Factors.”

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